Project Summary/Abstract Germ cells give rise to gametes?oocytes and sperm?that fuse to create a totipotent zygote, which divides and generates all the cells of an organism. During development, a new germ cell lineage must be established and differentiate properly to ensure propagation of the species. Most knowledge of germ cell specification and development comes from model organisms in which maternal determinants specify germ cells early in embryogenesis. By contrast, many animals (including mammals) specify their germ cells in response to inductive cues from surrounding cells later in development. Despite these differences in germline establishment, all germ cells share several important features, such as intrinsic transcriptional repression of somatic cell programs and dependence on extrinsic factors from somatic support cells. The free-living planarian flatworm, Schmidtea mediterranea, is well known for its extraordinary regenerative prowess, which relies on a population of pluripotent somatic stem cells. Planarians specify their germ cells post-embryonically from these somatic stem cells in response to inductive cues. Strikingly, planarians can even regenerate new germ cells de novo. Work over the past decade has established the planarian as a tractable model in which to study germ cell biology, and several intrinsic and extrinsic factors that regulate distinct aspects of male germline development?specification, maintenance, and differentiation?have been identified. However, almost nothing is known about female germ cell development in these animals, a critical gap because of the important role of egg production in the pathology of parasitic flatworm diseases. Here, building on extensive preliminary data from the applicant?s laboratory, the following two aims are proposed: (i) to functionally characterize the ovarian transcriptome; and (ii) to examine the role(s) of monoamine neurotransmitters in germ cell development. In Aim 1, laser capture microdissection and next-generation RNA sequencing will be used to create ovary- and testis-enriched transcriptomes. Validating ovary-enriched expression by in situ hybridization and characterizing gene function by RNA interference will enable the identification of germ cell-intrinsic and extrinsic regulators of female germ cell development. In Aim 2, cutting-edge mass spectrometry-based metabolomic studies will be combined with gene expression mapping and functional approaches to investigate the biogenic monoamines that signal locally and/or systemically to regulate germ cell development. The proposed experiments capitalize upon the planarian?s regenerative abilities and the functional genomic tools available for studying these animals to acquire a comprehensive view of the genes regulating female germ cell development. These studies are significant as they are expected to enhance understanding of germ cell biology across the metazoans and help guide future experiments on mammalian systems. In addition, this work will provide novel targets for disrupting reproduction in parasitic flatworms, which capitalize on their prolific reproductive output to propagate and afflict hundreds of millions of people worldwide.